Nucleoside transport plays an important role in cellular physiology. The existence of mutant cell lines which have lost their capacity to transport virtually all purine and pyrimidine nucleosides across the plasma membrane suggests a common transport function for all nucleosides in mammalian cells. The transport-deficient cells are resistant to nucleoside-mediated growth inhibitory effects. We propose to develop a system to analyze nucleoside transport in mammalian cells. Genetic studies will be performed on multiple human nucleoside transport-defective cells to confirm the specificity of this transport system. We have demonstrated genetically that the nucleoside transport function has a profound regulatory effect on nucleoside efflux and on the rates of de novo purine synthesis and purine salvage. Complementation analysis by cell fusion techniques will determine the number of genetic loci affecting transport function. A mapping system based on human-mouse cell hybrids will be developed to ascertain the chromosomal location of the human genes for nucleoside transport. Proteins from wild-type and mutant cells deficient in nucleoside transport will be compared by two-dimensional electrophoresis and by photoaffinity labeling in order to identify the relevant gene product(s). Competitive binding assays with photoaffinity ligands and purine and pyrimidine nucleosides will substantiate the biochemical identification of the nucleoside transporter. The photoaffinity-labeled nucleoside transport protein and an inhibitor binding assay will be used to monitor solubilization and purification by affinity chromatography. For further detailed characterization of the nucleoside transport functions in wild-type and mutant cells, immunological reagents will be generated. (P)